The present invention relates to the use of a new principle for
(a) improving pregnancy potential with ERxcex2-agonists in connection with in vivo treatment and in connection with in vitro fertilisation (IVF) and embryo transfer treatment and
(b) inhibiting pregnancy potential with ERxcex2-antagonists.
More specificially, the application relates to
(a) improvement of pregnancy potential of oocytes and preimplantational embryos by improvement and stimulation of ovarian folliculogenesis and
(b) inhibition of pregnancy potential by inhibiting folliculogenesis, ovulation and preimplantational development of ovulated oocytes.
This present invention relates further to new compounds as pharmaceutical active ingredients, which have in vitro a higher affinity to estrogen receptor preparations of rat prostates than to estrogen receptor preparations of rat uteri and exert a contraceptive action in vivo by their preferential action on the ovary, to their production, their therapeutic application and pharmaceutical forms for dispensing that contain the new compounds.
The chemical compounds are novel, steroidal, tissue-selective antiestrogens.
Follicular growth refers to the development of an ovarian follicle from the primordial to antral follicle. Patients suffering from ovarian infertility, e.g. polycystic ovarian syndrom patients, have a disordered folliculogenesis and the numbers of primary and secondary follicles are about twice those observed in the normal ovary (Hughesden (1982), Obstet Gynecol Survey 37:59-77). Therefore, it may be concluded that follicular growth is often impaired in infertility patients.
There is evidence that most, if not all, of the steps from primordial to early antral follicles are gonadotropin-independent but it is not yet clear which of the many candidates among the paracrine and autocrine factors which have been identified in preantral follicles are the most important for early follicular growth (Elvin et al. (1999), Mol Cell Endocrinol 13:1035-1048; McNatty et al. (1999), J Reprod Fertil Suppl 54:3-16). Gonadotropins are mainly involved for the late steps of folliculogenesis, e.g. the transition from an early antral follicle to an ovulatory follicle.
Couples seeking infertility treatment will often be subject to different in vivo and in vitro treatment regimes. In vitro and vivo infertility (IVF) treatment regimens often consists of ovulation induction by pretreatment with exogenous gonadotrophins, mainly follicle stimulating hormone (FSH) and antiestrogens (White et al. (1996), J Clin Endocrinol Metab 81:3821-3824). The in vitro treatment protocol then involves retrieving the oocytes from the preovulatory, antral follicles of the ovaries in order to be matured and/or fertilised in vitro. After fertilisation and preimplantational embryo development, one to three embryos are retransfered in the woman""s uterus in order to get a successful pregnancy. IVF is now an established treatment, which has been performed on a large scale for more than 20 years.
The protocols used for administration of exogenous gonadotropins are numerous and not without risks and disadvantages. The major disadvantages includes the risk of achieving ovarian hyperstimulation syndrome (OHSS) which in severe cases may be life threatening, the economic costs to the couple, side effects from the gonadotropin preparations including weight gain, bloating, nausea, vomiting, and the unknown long-term cancer risk.
One way to alleviate the risks, side effects, and economic disadvantages of controlled ovarian stimulation protocols would be to properly mature and stimulate follicular growth of ovarian infertility patients in vivo with a suitable agent before exogenous gonadotropin treatment starts. This approach would imply that the women are without gonadotropin stimulation or receive a minimal exogenous gonadotropin stimulation.
On the other hand contraceptive methods involving the administration of chemical substances are widely practiced among women who desire to limit pregnancies. Among the presently used chemical methods of fertility control, the most important are those which act by means of the following:
(a) suppression of ovulation through inhibition of gonadotropin release (inhibition of the endocrine principle),
(b) alteration of the female reproductive tract to prevent migration of sperm to the site of fertilisation,
(c) blocking the implantation of the zygote (nidation),
(d) spermicidal action or
(e) an abortifacient.
The oral contraceptives, consisting of an estrogen combined with a progestin or a progestin alone, are the most prominent chemical contraceptive agents. The contraceptives of the combined type act primarily by suppressing ovulation by negative feedback to prevent gonadotropin release by the pituitary. Although the oral contraceptives are highly effective, their use is associated with unpleasant side effects, such as nausea, depression, weight gain and headache, and an increased long-time risk of severe disease, such as thromboembolism, stroke, hepatic adenoma, gall bladder disease, hypertension and bleeding irregularities, indicating that an effective contraceptive principle without sides effects is presently not available. Therefore a need exists in medicine for new contraceptive methods.
An ideal contraceptive compound acting on the ovarian follicle would be an agent that does not modify the endocrine pituitaryxe2x80x94ovarian communication system but selectively interrupt folliculogenesis, e.g. the paracrine interplay between the granulosa cell and the oocyte, leading
(a) to an impairment of the follicular programm and an ovulated oocyte that is uncapable to get fertilised or
(b) to an impairment of the follicular programm and an inhibition of ovulation,
Follicular growth is the development of an ovarian follicle from the primordial stage to the large antral follicle that is ready to burst. Only an optimally built-up antral follicle has the potential to ovulate a mature egg cell. Patients with ovarian infertility, e.g., PCOS (=polycystic ovarian syndrome) patients, have a disrupted folliculogenesis associated with hormonal and ovulation disorders as well as insufficiently matured egg cells (Franks et al. (2000) Mol Cell Endocrinol 163:49-52).
There are always more indications that the early stages of folliculogenesis, i.e., the development steps from the primordial follicle to the early antral follicle, are gonadotrophin-independent, but it is still not conclusively explained which of the identified autocrine or paracrine factors (Elvin et al. (1999). Mol Cell Endocrinol 13:1035-1048; McNatty et al. (1999), J Reprod Fertil Suppl 54; 3-16) are the most important in early folliculogenesis. Gonadotrophins, such as, e.g., FSH (follicle-stimulating hormone), however, are mainly involved in the late steps of folliculogenesis, i.e., the development from the early antral follicle to the large ovulatory follicle. Additional modulators of folliculogenesis are also discussed in the late folliculogenesis, however (Elvin et al. (1999), Mol Cell Endocrinol 13:1035-1048).
Until recently, only a single type of estrogen receptor (ER) has thought to exist and mediate the genomic effects of 17xcex2-estradiol in mammalian tissues. However, the cloning of a gene encoding a second type of ER, termed ERxcex2 has prompted a reevaluation of estrogen actions (Kuiper et al (1996), Proc Natl Acad Sci 93:5925-5930). Whereas transcripts encoding the classical ER (now referred to as ERxcex1) are detected in nearly all tissues assayed from both sexes in animal models, the highest expression of ERxcex2 mRNA can be observed in the ovary (Couse et al. (1997), Endocrinology 138:4613-4612). Because ERxcex1 and ERxcex2 have different tissue distributions, especially regarding the ovary, differences in ligand interaction or activity with the two ERs could translate into important differences in their biological actions at the tissue level. After the successful generation of ERxcex1 and ERxcex2 knockout mice (Couse et al. (1999), Endocrine Reviews 20:358-417) it is now known that ERxcex1 and ERxcex2 mediate different actions in the female reproductive tract and that ERxcex1 plays an important role in the function of the adult uterus, the mammary gland, in the negative regulation of gonadotropin action, whereas ERxcex2 is mainly involved in ovarian physiology.
ERxcex1 and ERxcex2 have significantly different primary sequences in their ligand binding and transactivation domains. This suggests that these ER subtypes might bind some ligands with different affinity and that these ligands might also have different agonist or antagonist character mediated by the two receptors.
Recent patent applications (WO 00/47503, WO 00/63228, PCT/EP00/10804, DE 100 19 167.3 and U.S. 60/207,370) and publications (Sun et al. (1999), Endocrinology 140:800-804; Stauffer et al. (2000), J Comb Chem 2:318-329) showed that high-affinity steroidal and nonsteroidal ligands for the ERxcex1 and ERxcex2 were found. Some compounds were considerably more potent as agonists/antagonists on the ERxcex1 where others were considerably more potent as agonists/antagonists on the ERxcex2. In WO 00/31112 new steroid compounds based on estradiol and carrying a hydrocarbon as 11xcex2-substitution which has one single linear chain having a length of from 5 to 9 carbon atoms. The compounds have an ERxcex1-agonist/ERxcex2-antagonist profile. The mixed estrogen-receptor profile makes these compounds suitable as improved estrogens for treatment of estrogen-related disorders and in contraception together with a progestogen.
All above mentioned applications and publications are incorporated herein by reference
However, so far a selective action of ER subtype-specific interaction in vivo could not be shown.
The present invention shows for the first time the effects of ERxcex2 agonists/antagonists in defined animal models. The data obtained are the first showing that ERxcex2 subtype-specific compounds are useful in defining biological activities in the ovary. From these data it becomes evident that ERxcex2 plays an important role in
(a) early folliculogenesis and
(b) ovulation which can be taken as a new principle for treatment of infertility and the design of new contraceptives.
The following examples serve for explaining the invention without limiting the invention to these examples.
Intact immature female Wistar rats were hypophysectomized (hypex) on day 24 of age (d0). The following hormone treatments were initiated six days after surgery:
(a) hypex rats treated with vehicle (control group) (ethanol/arachisoil: 1+9 [v/v]) only
(b) hypex rats treated with 0.01, 0.1 and 1 mg 17xcex2-estradiol/day (compound A)
(c) hypex rats treated with 0.01, 0.1 and 1 mg of an ERxcex1-selective, 3,17xcex2-Dihydroxy-19-nor-17xcex1-pregna-1,3,5(10)-triene-21,16xcex1-lactone (compound B, DE 100 48 634.7);
(d) hypex rats treated with 0.01, 0.1 and 1 mg of an ERxcex2-selective compound #1,8xcex2-Vinyl-1,3,5(10)-estratriene-3,17xcex2-diol (compound C, DE 100 19 167.3/U.S. 60/207,370);
(e) hypex rats treated with 0.01, 0.1 and 1 mg of an ERxcex2-selective compound #2 (16a-Hydroxy-9a-methyl-estra-1,3,5(10)-triene, compound D).
Animals were subcutaneously treated once per day for 4 consecutive days with the respective compounds or vehicle (day 6, 7, 8, 9). Number of animals per group were 5-6. The animals were randomly assigned to the treatment and vehicle (control) group.
One day after the last injection (day 10) animals were weighed and sacrificed by carbon dioxide asphyxiation. The ovaries were excised and weighed. Relative ovarian weights (in milligrams), defined as relative ovarian wet weights, were calculated per 100 g body weight for each animal. For each treatment group and control group the mean relative wet weights were determined. The one way ANOVA was applied to the data in order to compare treatment group values with the respective control group.
For histological evaluation, ovaries were placed in neutral buffered 3.7% formaldehyde solution. The ovaries were then embedded in paraffin, cut into 4 xcexcm transverse sections, and stained with hematoxylin and eosin. Ovarian sections were then evaluated qualitatively for folliculogenesis.
The results presented in FIG. 1 demonstrate that 17xcex2-estradiol, which is a ligand to ERxcex1 as well as to ERxcex2, and both ERxcex2-specific ligands dose-dependently are able to significantly increase ovarian wet weight. In contrast, the ERxcex1-specific ligand was not able to stimulate ovarian growth.
The histological data presented in FIG. 2 clearly demonstrate that, compared to control animals, the increase in ovarian weight after treatment with 17xcex2-estradiol and the ERxcex2-specific ligands are due to an enlargement in follicular size. Only a few small follicles are found. After treatment with the ERxcex1-specific ligand the ovaries exhibit many small atretic follicles comparable to the vehicle control group.
Effect of an ERxcex2-selective Antagonist an Ovulation Rate in Immature Rat
Superovulation experiments were carried out in three groups of intact immature female Wistar rats on day 23 of age. Number of animals per group were 5. The animals were randomly assigned to the three groups.
Each animal received a single subcutaneous (s.c.) injection of 20 I.U. pregnant mare""s serum gonadotropin (PMSG) at day 0. At the same time (day 0) as well as 24 hours and 48 hours later the animals were s.c. injected with
(a) the vehicle formulation (control group: ethanol/aracisoil: 1+9 [v/v]) (group 1) or
(b) an ERxcex2- selective antagonist 17-Chloro-17axcex1-(trifluoromethyl)-17a-homoestra-1,3,5(10)16-tetraene-3,17axcex2-diol (compound E) at the dose of 1 mg/kg (group 2) and 10 mg/kg (group 3). 54 hours after the start of the experiment the animals got a single intraperitoneal (i.p.) injection of 10 I.U. human chorion gonadotropin (hCG). The animals were then killed 16 hours after the hCG injection.
The ovaries and oviducts were removed. The ovulated oocyte/cumulus mass was extracted from the oviduct by flushing the oviduct with M-2 medium and the oocytes per animal were counted after enzymatic disassociation from the surrounding cumulus with M-2 medium supplemented with 0.3% hyaluronidase.
The results presented in Table 1 demonstrate that the ERxcex2-specific antagonist was dose-dependently able to decrease ovulation rate from 13.2 in the group 1 (control) to 8.0 in group 3.
Effect of an ERxcex2-selective Ligand on Late Folliculogenesis and Ovulation in Hypophysectomized Rats
Intact immature female Wistar rats were hypophysectomized (hypex) on day 24 of age (day 0). The following hormone treatments were initiated six days after surgery:
(a) hypex rats treated with vehicle (control group) (ethanol/arachisoil: 1+9 [v/v]) only
(b) hypex rats treated with 0.01, 0.1 and 1 mg 17xcex2-estradiol/day
(c) hypex rats treated with 0.01, 0.1 and 1 mg of the ERxcex1-selective compound B
(d) hypex rats treated with 0.01, 0.1 and 1 mg of the ERR-selective compound C
Animals were subcutaneously treated once per day for 4 consecutive days with the respective compounds or vehicle (day 6, 7, 8, 9). Number of animals per group were 57. The animals were randomly assigned to the treatment and vehicle (control) group.
One day after the last injection (day 10) animals were subcutaneously injected with 20 IU prgenant mare serum gonadotropin (PMSG). Two days later, at day 13, 10 IU human Chorionic Gonadotropin (hCG) were injected intraperitoneally in order to induce ovulation. At day 14 animals were weighed and sacrificed by carbon dioxide asphyxiation. The oviducts and ovaries were removed:
The oviducts were flushed with a M-2 medium and the oocytes per animal were counted after enzymatic disassociation from the surrounding cumulus with M-2 medium supplemented with 0.3% hyaluironidase. For each treatment group and control group the mean number of ovulated oocytes were calculated.
The ovaries were excised and weighed. Relative ovarian weights (in milligrams), defined as relative ovarian wet weights, were calculated per 100 g body weight for each animal. For each treatment group and control group the mean relativewet weights were determined.
The one way ANOVA was applied to the data in order to compare treatment group values with the respective control group.